Characteristics and Commercial Utility of Starch
Starch is a mixture of two polysaccharides, amylose and amylopectin. Amylose is an unbranched chain of up to several thousand xcex1-D-glucopyranose units linked by xcex1-1,4 glycosidic bonds. Amylopectin is a highly branched molecule made up of up to 50,000 xcex1-D-glucopyranose residues linked by xcex1-1,4 and xcex1-1,6 glycosidic bonds. Approximately 5% of the glycosidic linkages in amylopectin are xcex1-1,6 bonds, which leads to the branched structure of the polymer.
Amylose and amylopectin molecules are organized into granules that are stored in plastids. The starch granules produced by most plants are 15-30% amylose and 70-85% amylopectin. The ratio of amylose to amylopectin and the degree of branching of amylopectin affects the physical and functional properties of the starch. Functional properties, such as viscosity and stability of a gelatinized starch determine the usefulness and hence the value of starches in food and industrial applications. Where a specific functional property is needed, starches obtained from various crops such as corn, rice, or potatoes may meet the functionality requirements. If a starch does not meet a required functional property, if for example it must have stable viscosity under high temperatures and acidic conditions, the functionality can sometimes be achieved by chemically modifying the starch. Various types and degrees of chemical modification are used in the starch industry, and the labeling and use of chemically modified starches must meet government regulations.
Within the starch bearing organs of plants, the proportion of amylose to amylopectin and the degree of branching of amylopectin are under genetic control. For example, plants homozygous recessive for the waxy (wx) gene lack a granule-bound starch synthase enzyme and produce nearly 100% amylopectin. Plants homozygous recessive for the amylose extender (ae) gene can produce starch granules that are up to 90% amylose (see U.S. Pat. No. 5,300,145). The dull gene has been shown to influence the levels of activity of a starch synthase and a starch branching enzyme.
Most cereal crops are handled as commodities, and many of the industrial and animal feed requirements for these crops can be met by common varieties which are widely grown and produced in volume. However, there exists at present a growing market for crops with special end-use properties which are not met by grain of standard composition. Most commonly, specialty corn is differentiated from xe2x80x9cnormalxe2x80x9d corn, also known as field corn, by altered endosperm properties, such as an overall change in the ratio of amylose to amylopectin as in waxy or high amylose corn, an increased accumulation of sugars as in sweet corn, or an alteration in the degree of endosperm hardness as in food grade corn or popcorn; Glover, D. V. and E. T. Mertz, (1987) in Corn: Nutritional Quality of Cereal Grains; Genetic and Agronomic Improvement, R. A. Olson and K. J. Frey, eds. American Society of Agronomy, Madison Wisconsin, pp. 183-336. Rooney, L. W. and S. O. Serna-Saldivar, (1987) Food Uses of Whole Corn and Dry-milled Fractions, in Corn: Chemistry and Technology, S. A. Watson and P. E. Ramstead, eds. American Association of Cereal Chemists, Inc., St. Paul, Minn., pp. 399-429. The current invention offers the buyers of specialty corn a source of starch having properties distinct from waxy starch and offers farmers the opportunity to grow a higher value-added crop than normal or waxy corn.
Purified starch is obtained from plants by a milling process. Corn starch is extracted from kernels through the use of a wet milling process. Wet milling is a multi-step process involving steeping and grinding of the kernels and separation of the starch, protein, oil and fiber fractions. A review of the corn wet milling process is given by S. R. Eckhoff in the Proceedings of the Fourth Corn Utilization Conference, Jun. 24-26, 1992, St. Louis, Mo., printed by the National Corn Growers Association, CIBA-GEIGY Seed Division and the United States Department of Agriculture. Starch is used in numerous food and industrial applications and is the major source of carbohydrates in the human diet. Typically, starch is mixed with water and cooked to form a thickened gel. Three important properties of a starch are the temperature at which it cooks, the viscosity the gel reaches, and the stability of the gel viscosity over time. The physical properties of unmodified starch during heating and cooling limit its usefulness in many applications. As a result, considerable effort and cost is needed to chemically modify starch in order to overcome these limitations of starch and to expand the usefulness of starch in industrial applications.
Some limitations of unmodified starches and properties of modified starches are given in Modified Starches: Properties and Uses, O. B. Wurzburg, ed., (1986) CRC Press Inc., Boca Raton, Fla. Unmodified starches have very limited use in food products because the granules swell and rupture easily, thus forming weak bodied, undesirable gels. Chemical modifications are used to stabilize starch granules thereby making the starch suitable for thousands of food and industrial applications including baby foods, powdered coffee creamer, surgical dusting powders, paper and yarn sizings and adhesives. Common chemical modifications include cross linking in which chemical bonds are introduced to act as stabilizing bridges between starch molecules, and substitution in which substituent groups such as hydroxyethyl, hydroxypropyl or acetyl groups are introduced into starch molecules.
The use of chemically modified starches in the United States is regulated by the Food and Drug Administration (FDA). xe2x80x9cFood starch-modifiedxe2x80x9d starches may be used in food but must meet specified treatment limits, and xe2x80x9cindustrial starch-modifiedxe2x80x9d starches may be used in items such as containers that come in contact with food and must also meet specified treatment requirements; Code of Federal Regulations, Title 21, Chapter 1, Part 172, Food Additives Permitted in Food for Human Consumption, Section 172, 892, Food Starch-Modified, U. S. Government Printing Office, Washington, D. C. 1981; (a) Part 178, Indirect Food Additives, Sect. 178.3520, Industrial Starch-Modified. These regulations limit the degree of chemical modification by defining the maximum amount of chemical reagent that can be used in the modification steps. The levels of by-products in starch resulting from the modification process are also regulated. For example, propylene chlorohydrin residues in hydroxypropyl starch are of special concern; Tuschhoff, J. V., (1986) Hydroxypropylated Starches, In Modified Starches: Properties and Uses, O. B. Wurzburg, ed., CRC Press, Boca Raton, Fla. pp. 89-96.
Alteration of Starch Fine Structure Through Molecular Genetic Manipulation of Starch-Bearing Plants
Differences in the degree of starch branching or polymerization are known to result in a change in the physiochemical properties of starch. It has been suggested that starches, tailor-made for specific applications, may be generated by alteration of the branch chain distribution of the amylopectin molecule, the relative proportion of amylose to amylopectin or the degree of polymerization of amylose. However, achieving phenotypic alteration of starch composition has been problematic; while key enzymes in starch biosynthesis have been identified, their exact roles remain uncertain. Thus, correlation of activities of particular enzymes with particular molecular characteristics of starch structure and, in turn, with starch function in food and industrial products has been difficult. Although desirable functional properties that an ideal starch might need can be envisioned, there is only a vague understanding of what the molecular structure of the starch should be to achieve this and little understanding of how particular starch biosynthetic enzymes specifically affect those parameters. For example, the role of individual enzymes in determining the branching patterns and length of branches is as yet unclear and is compounded by the lack of understanding of how branching enzymes and starch synthases interact.
WO 94/09144 discusses the generation of plants with improved ability to synthesize starch at elevated temperatures. This publication proposes that the limiting factor in grain filling at elevated temperature is the lability of certain starch biosynthetic enzymes, particularly starch synthase (SS) and starch branching enzyme (SBE). The introduction of genes encoding enzymes that have a higher optimum temperature for activity or that have a higher tolerance to heating into plants may afford an increase in the amount of starch deposited in the corn kernel. Moreover, it is claimed that this strategy may be used to generate starch of altered fine structure as a result of the introduction of donor genes whose expression may alter the balance of the different starch biosynthetic enzymes. Suggested donor genes include those that encode enzymes that display improved kinetic or allosteric properties relative to the endogenous enzyme or an extra copy of the endogenous gene that would compensate for losses in enzyme activity incurred due to heat lability. As a means to alter starch structure, WO 94/09144 also suggests the use of sense and antisense genes to alter the natural ratios of the different starch synthase and branching enzymes in the recipient plant. This publication discloses the effect of temperature on catalytic activity and enzyme stability for certain starch biosynthetic enzymes, however, no data are presented to subsantiate the proposed molecular strategies.
The results of attempts to inhibit SBE expression in potato using an antisense approach were recently reported by Virgin et al. at the 4th International Congress of Plant molecular Biology (June, 1994) and by Christensen et al. and Kossman et al. at the Plant Polysaccharide Symposium (July, 1994). In all cases, although SBE activity was almost completely abolished, the amylose-to-amylopectin ratio remained unaltered. Both Virgin et al. and Kossman et al. reported no change in amylopectin structure. However, Christensen et al. did report a change in the distribution of branch chains on the amylopectin molecule with an increase in the number of long branches.
The results in potato are unexpected, since only a single starch branching enzyme has been purified and only a single gene has been detected on Southern blots of potato genomic DNA, even under conditions of low stringency (Kooshnoodi, J. et al. (1993) FEBS Letters 332:132-138; Kossman, J. et al. (1991) Mol. Gen. Genet. 230:39-44). Thus, antisense suppression of the single starch branching enzyme gene in potato, resulting in significant reduction of enzyme levels and a concomitant decrease in starch branching enzyme activity, was expected to result in a measurable, reproducible change in starch composition and starch fine structure. The contrary and inconsistent results reported in the literature suggest that other starch branching enzyme genes that share little homology with the identified gene may also play a role in determining amylopectin structure in potato. Alternatively, branching enzyme activity in potato may be encoded by a single gene, but the protein may be present in such large excess that amylopectin quantities or structure are not affected even when greater than 90% of the enzyme activity is inhibited.
Alteration of starch fine structure in corn is complicated by the fact that three SBE isoforms have been identified. In corn endosperm, the three isoforms that demonstrate starch branching enzyme activity are SBEI, SBEIIa and SBEIIb. In the amylose extender (ae) mutant, SBEIIb activity has been found to be deficient while in the dull (du) mutant, decreased levels of SBEIIa are observed (Boyer, C. D. and Preiss, J. (1981) Plant Physiol. 67:1141-1145). Studies of the catalytic properties of the corn starch branching enzymes indicate that the isoforms differ in substrate preference and in the length of glucan chain that is transferred. SBEI activity is higher when amylose serves as the substrate, and longer chains are preferentially transferred. The SBEII isoforms display higher activity with more highly branched substrates such as amylopectin. These enzymes preferentially transfer shorter glucan chains (Guan et al. (1993) Plant Physiol. 102:1269-1273; Takeda et al. (1993) Carbohydrate Res. 240:253-263).
A corn SBEI cDNA has been cloned and sequenced (Baba et al. (1991) Biochem. Biophys. Res. Commun. 181:87-94; Fisher et al. (1995) Plant Physiol. 108:1313-1314). In addition, a corn SBEII cDNA clone has been isolated and the nucleotide sequence of the clone has been published (Fisher et al. (1993) Plant Physiol. 102:1045-1046). This cDNA clone maps to the ae locus, confirming that this locus encodes the structural gene for corn SBEIIb (Stinard et al. (1993) Plant Cell 5:1555-1566).
Starch isolated from the ae mutant is known to differ in structure from that isolated from dent corn (Baba et al. (1984) Agric. Biol. Chem. 48:1763-1775). The effect of the ae allele on starch properties has been investigated (Yamada et al. (1978) Starke 30:145-148). Increasing doses of ae in a waxy (wx) background produce an increase in the gelatinization temperature so that for the homozygous mutant, incomplete cooking of the starch is observed, even at 95xc2x0 C. These authors indicate that the increase in viscosity associated with ae wx starch is highly desirable and suggest a xe2x80x9ctargetxe2x80x9d starch with properties intermediate between wx and ae wx. While mutations which influence the levels of corn SBEIIa and SBEIIb are available, mutations in the SBEI structural gene have yet to be identified. The lack of SBEI mutants may indicate that the absence of this branching enzyme isoform is lethal to the plant. Alternatively, a SBEI null mutation may give rise to no observable change in seed phenotype or one that is not readily distinguished from existing starch mutants.
Molecular genetic solutions to the generation of starches from corn with altered fine structures have a decided advantage over more traditional plant breeding approaches. Changes to starch fine structure can be produced by specifically inhibiting expression of one or more of the SBE isoforms by antisense inhibition or cosuppression. An antisense or cosuppression construct would act as a dominant negative regulator of gene activity. While conventional mutations can yield negative regulation of gene activity these effects are most likely recessive. The dominant negative regulation available with a transgenic approach may be advantageous from a breeding perspective. Additionally the ability to restrict the expression of the altered starch phenotype to the reproductive tissues of the plant by the use of specific promoters may confer agronomic advantages relative to conventional mutations which will have an effect in all tissues in which the mutant gene is ordinarily expressed. Finally, the variable levels of antisense inhibition or cosuppression that arise from chromosomal position effects could produce a wider range of starch phenotypes than those that result from dosage effects of a mutant allele in corn endosperm.
The complex organization of starch branching enzymes in corn endosperm and the results reported in potato render attempts to manipulate starch fine structure by inhibition of gene expression of one of the known corn isoforms unpredictable. Reported scientific evidence indicates that inhibition of expression of a single starch branching enzyme gene and a measurable reduction of starch branching enzyme activity is not predictive of a corresponding change in starch fine structure. Moreover, antisense technology is inherently uncertain in that it is not obvious or predictable whether the entire gene or whether specific fragments and which fragments of a gene will be most effective in mediating strong antisense inhibition. Some results do indicate that strong expression of the antisense gene is required; however, good expression of the antisense transcript does not necessarily correlate with the observation of and the strength of the antisense phenotype (Bourque, J. (1995) Plant Sci. 105:125-149). Although several theories have been advanced to explain the phenomenon of cosuppression (Flavell, R. B. (1994) Proc. Natl. Acad. Sci. (USA) 91:3490-3496), it has become apparent that no single mechanism appears sufficient to describe all of the observed results. To date, cosuppression effects have been reported in tobacco, canola, soybean, tomato and Arabidopsis, all of which are dicot plants. No data have been reported that indicates that this phenomenon is operable in monocots.
Notwithstanding the ability to inhibit the expression of SBE genes in corn, a resulting change in starch phenotype remains unpredictable. Although the enzymatic steps are known, the molecular details of starch biosynthesis are not well understood. It is not clear whether the three SBE isoforms contribute equally throughout starch biosynthesis or whether each isoform plays a distinct role in assembling the amylopectin molecule at discrete steps along an obligatory pathway. In consideration of the possible interplay between the starch branching enzymes and the multiple starch synthases that function in glucan chain elongation, it is impossible to make predictions concerning starch structure based upon the catalytic properties of each isoform.
The instant invention discloses utilization of a cDNA clone to construct sense and antisense genes for inhibition of starch branching enzyme enzymatic activity in corn grain or endosperm. More specifically, this invention concerns a method of controlling the branch chain distribution of the amylopectin, the relative proportion of amylose to amylopectin and the degree of polymerization of amylose components of starch in corn comprising: (1) preparing a chimeric gene comprising a nucleic acid fragment encoding a starch branching enzyme structural gene or a fragment thereof, operably linked in either sense or antisense orientation on the upstream side to a nucleic acid fragment that encodes a promoter that directs gene expression in corn endosperm tissue, and operably linked on the downstream side to a nucleic acid fragment encoding a suitable regulatory sequence for transcriptional termination, and (2) transforming corn with said chimeric gene, wherein expression of said chimeric gene results in alteration of the branch chain distribution of the amylopectin molecular component of starch derived from the grain of said transformed corn compared to the branch chain distribution of the amylopectin molecular component of starch derived from corn not possessing said chimeric gene. This invention also concerns corn varieties prepared by transformation using said method, starch isolated from the grain of a corn variety prepared using said method, and a method of preparing a thickened foodstuff comprising combining a foodstuff, water, and an effective amount of a starch isolated from the grain of a corn variety prepared using the said method, and cooking the resulting composition as necessary to produce said thickened foodstuff.